Cellular fluorescence emission spectra from two photon excitation of both added fluorophores (e.g. Indo-1) and from intrinsic fluorophores were measured using a cooled CCD detector and spectrometer. Induced fluorescence in cells complicates calibration of ratioed dyes, such as Indo-1, and therefore needs to be characterized. In addition, in vivo fluorescence emission spectra may be a sensitive indicator of cellular environmental characteristics. Using two photon excitation, the measured emission spectrum arises only from the focal volume, which is typically less than one cubic micron and provides for optimal spatial resolution. In addition, this technique works well in thick tissues such as the dermis of human skin where the in vivo spectrum of elastin was measured and will allow for the spatial characterization of in vivo fluorescence from structures such as intact tumors or other pathologies.